RESEARCH ARTICLE

Inhibition of itch by neurokinin 1 receptor(Tacr1) -expressing ON cells in the rostral
ventromedial medulla in mice
Taylor Follansbee1,2*, Dan Domocos3, Eileen Nguyen4, Amanda Nguyen1,
Aristea Bountouvas1, Lauren Velasquez1, Mirela Iodi Carstens1, Keiko Takanami5,Sarah E Ross4, Earl Carstens1
1

Department of Neurobiology, Physiology and Behavior, University of California,
Davis, Davis, United States; 2Department of Neuroscience, Johns Hopkins University,
Baltimore, United States; 3Department of Anatomy, Animal Physiology andBiophysics, University of Bucharest, Bucharest, Romania; 4Pittsburgh Center for Pain
Research and Department of Neurobiology, University of Pittsburgh, Pittsburgh,
United States; 5Department of Environmental Life Science, National Nara Women
University, Nara, Japan*For correspondence:
tfollansbee@ucdavis.edu
Competing interest: The authors
declare that no competinginterests exist.
Funding: See page 14
Preprinted: 06 April 2021
Received: 21 April 2021
Accepted: 04 August 2022
Published: 16 August 2022
Reviewing Editor: Rohini Kuner,
Heidelberg University, Germany‍ ‍Copyright Follansbee et al.
This article is distributed under
the terms of the Creative
Commons Attribution License,
which permits unrestricted use
and redistribution provided thatthe original author and source
are credited.

Abstract The rostral ventromedial medulla (RVM) is important in descending modulation of
spinal nociceptive transmission, but it is unclear if the RVM also modulates spinal pruriceptive transmission. RVM ON cells are activated by noxious algesic and pruritic stimuli and are pronociceptive.
Many RVM-­spinal projection neurons express the neurokinin-­1 receptor (Tacr1), and ON-­cells are
excited by local administration of substance P (SP). We hypothesized that Tacr1-­expressing RVM ON
cells exert an inhibitory effect on itch opposite to their pronociceptive action. Intramedullary microinjection of SP significantly potentiated RVM ON cells and reduced pruritogen-­evoked scratching
while producing mild mechanical sensitization. Chemogenetic activation of RVM Tacr1-­expressing
RVM neurons also reduced acute pruritogen-­evoked scratching. Optotagging experiments
confirmed RVM Tacr1-­expressing neurons to be ON cells. We conclude that Tacr1-­expressing ON
cells in RVM play a significant role in the modulation of pruriceptive transmission.
Editor's evaluation
This manuscript uses several complementary strategies to investigate and manipulate the activity ofneurokinin 1 receptor-­expressing neurons in the rostroventral medulla of mice in the context of itchsensation. The study delivers important insights into supraspinal itch processing and descending
modulation of itch signals in the spinal cord.

Introduction
The transmission of somatosensory information in the spinal cord is under top-­down modulationand has been extensively studied in the context of pain signaling. Descending modulation of pain isreflected by phenomena such as expectation (placebo and nocebo), diffuse noxious inhibitory control(DNIC), and conditioned pain modulation (CPM) (Bartels et al., 2018; Chebbi et al., 2014; Damien
et al., 2018; Le Bars, 2002; Lockwood and Dickenson, 2020). Descending modulation is also
thought to underlie stress mediated changes in pain threshold, with acute stress inhibiting and chronic
stress facilitating nociceptive transmission (Butler and Finn, 2009; Fields, 2000; Jennings et al.,2014; Wager and Atlas, 2015). Recent studies indicate that the spinal transmission of pruriceptive

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information is also under descending modulatory influences, but it is not known whether itch is modulated in the same way as pain (Agostinelli and Bassuk, 2021; Gao et al., 2021; Gao et al., 2019;
Koga et al., 2020; Liu et al., 2019; Samineni et al., 2019; Wu et al., 2021).
The rostral ventromedial medulla (RVM) contains neurons with descending projections to thespinal cord which bidirectionally modulate spinal nociceptive transmission (Fields, 2000; Fields andBasbaum, 1978; Fields and Heinricher, 1985; Heinricher et al., 2009; Millan, 2002; Ossipov et al.,2014). RVM ON cells are excited and OFF cells are inhibited by noxious stimulation just prior to a
nocifensive withdrawal reflex, and respectively facilitate and inhibit spinal nociceptive transmission(Fields, 2004; Fields et al., 1983). Increased ON cell and decreased OFF cell firing are thought tocontribute to the chronification of pain (Ossipov et al., 2014). Neutral cells do not exhibit any significant response to noxious stimulation (Barbaro et al., 1986). Many RVM neurons project to the spinal
cord via the dorsolateral funiculus (DLF), terminating in layers I, II, and V, and are implicated in theanalgesic effects of opioids. It is still unknown whether the RVM ON and OFF cells are involved in themodulation of itch transmission.
A recent study reported that neurons in the periaqueductal gray (PAG) expressing Tac1, the genefor substance P (SP), project to and make an excitatory glutamatergic connection with neurons in
the RVM (Gao et al., 2019). Activation of these PAG neurons promotes scratching. A populationof GABAergic neurons originating in the RVM synapse onto spinal neurons that express the gastrin
releasing peptide receptor (GRPR) (Liu et al., 2019), which are considered to be essential for spinal
pruriceptive transmission (Sun and Chen, 2007). Activation of GABAergic and inhibition of glutamatergic neurons in PAG reduced scratching behavior in both acute and chronic itch conditions(Samineni et al., 2019). Furthermore, pruritogens were shown to excite ON and inhibit OFF cells(Follansbee et al., 2018). Cervical cold block, which diminishes activity in descending modulatory
pathways, decreased pruritogen-­evoked activity and facilitated algogen-­evoked activity of spinal cord
neurons implying opposing effects of descending modulatory pathways on nociceptive and pruriceptive transmission (Carstens et al., 2018). The current evidence suggests that at least two classes of
PAG neurons, via connections to RVM cells, exert bimodal effects on the spinal pain and itch signaling
pathways. Since activation of PAG tac1r neurons facilitates itch this implies that activation of RVM
Tacr1-­expressing neurons may likewise facilitate itch. However, RVM ON cells are known to facilitate
pain, and thus may have an opposing effect on itch transmission.
Here we investigated the role of Tacr1-­expressing RVM neurons in itch modulation. In the present
study, we used pharmacological and opto/chemogenetic methods to selectively activate RVM Tacr1-­
expressing cells, which we hypothesize represent a population of RVM ON cells, and assessed theeffects on pruritogen-­evoked scratching, and thermal and mechanical nociceptive behavior. When
RVM Tacr1-­expressings cells were activated, we observed that itch related behaviors were inhibited,while producing mild mechanical sensitization. We used optotagging to characterize these RVM Tacr1-­
expressing neurons as RVM ON cells. These results are the first to demonstrate an inhibitory effect of
RVM ON cells on itch transmission, contrary to their known pronociceptive function.

Results
SP enhances RVM ON-cell responses to pinch
In rats, responses of RVM ON cells were potentiated by intramedullary microinjection of the Tacr1
agonist SP (Budai et al., 2007) and we wished to determine whether this is true in mouse. We
identified RVM ON cells using in vivo extracellular electrophysiological recordings, with the criterion that noxious pinch elicited a >30% increase in firing that preceded the onset of the hindpaw
withdrawal. Using a microinjection cannula attached to a recording microelectrode (Figure 1A),
SP or saline was microinjected while recording from an ON cell in anesthetized mice. Microinjection of SP potentiated pinch-­evoked responses (Figure 1B). Following SP microinjection, normalized responses of ON cells to repeated pinch stimuli were significantly increased (Figure 1C). The
enhancement of responses lasted >1 hr. Histologically recovered recording sites were locatedwithin the RVM and adjacent regions of the medullary reticular formation (Figure 1D). These
results show that, similar to rats, RVM ON cells in the mouse are potentiated following localized
injection of SP.

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Figure 1. Effects of intramedullary microinjection of SP on RVM ON cells and itch and pain behavior. (A) SP or saline was microinjected while recordingfrom single ON cells. (B) Peristimulus-­time histogram of ON cell response to pinch before (left) and 5 min after local microinjection of SP (right).
(C) Normalized firing rate of ON cells following local microinjection of saline (blue, n=8) or SP (red, n=8) at time indicated by arrow. ON cells showeda significant increase in evoked firing following SP injection compared with saline (*; two-­way ANOVA, F1, 14 = 8.020, p=0.0133, bolded lines: mean
responses; error bars SEM). Male mice were used in these experiments. (D) Lesion sites from the RVM ON cell recordings. Numbers to right indicate
Bregma coordinates. (E) An implanted intramedullary microinjection cannula allowed assessment of itch and pain behavior after injection of SP into
RVM. (F, G) Graphs plot the number of scratch bouts elicited by intradermal injection of histamine (F, n=9, M = (106.2; 59.72), t=3.4113, DF = 8, 95% CI[15.06, 77.83], p=0.0092) or chloroquine (G, n=9, M = (102.9; 22.00), t=2.847, DF = 8, 95% CI [15.39, 146.5], p=0.0216) for each mouse (red dots andlines), and mean scratch bouts (black line; error bars: SEM), following intramedullary microinjection of saline or SP. Experiments with saline and SP
microinjections were conducted at least 7 days apart. Microinjection of SP significantly attenuated histamine- and chloroquine-­evoked scratching (F,G). (H) Mechanical withdrawal thresholds were reduced by intramedullary SP, n=10, M = (4.855; 3.253), t=8.96, DF = 8, 95% CI [1.198, 2.007], p = <0.0001(I) Thermal withdrawal latency was not significantly affected by intramedullary SP, n=8, M = (5.271; 4.691), t=0.9784, DF = 7, 95% CI [–0.8216, 1.981],
p=0.3605. (G, F) students t-­test *p<0.05, **p<0.01, ***p<0.001. n=5–7 males, 3 females/ group.
The online version of this article includes the following source data for figure 1:
Source data 1. Data for electrophysiolgical recordings and behavior from SP injection into the RVM.

Intramedullary SP inhibits scratching
Since RVM ON cells are potentiated following injection of SP, we next tested if intramedullary microinjection of SP affected itch and pain related behaviors. Mice were implanted with an intramedullary
microinjection cannula dorsal to the RVM to allow microinjection of SP or vehicle (Figure 1E). The
number of scratch bouts elicited by intradermal injection of histamine was significantly lower following
intramedullary microinjection of SP compared to saline vehicle (Figure 1F). Scratching elicited byintradermal injection of chloroquine was also significantly reduced following intramedullary injectionof SP compared to saline (Figure 1G).
There was a significant decrease in mechanical force to elicit a hindlimb withdrawal following intramedullary SP compared to vehicle injection (Figure 1H), indicating mild mechanical sensitization.
There was no significant effect of intramedullary SP injection on thermal hindpaw withdrawal latency(Figure 1I). We observed that both male and female mice showed a reduced hindlimb withdrawal,
but that males had a significantly higher force for both saline and SP injection when compared withfemales (Figure 3—figure supplement 3D). Thus, activation of RVM Tacr1-­
expressing neurons,
through intramedullary injection of SP, resulted in facilitation of mechanical nociceptive behavior andinhibition of pruritogen-­evoked scratching behavior.

Targeted expression of DREADDs in RVM Tacr1 expressing neurons
We next wanted to determine whether activation of RVM Tacr1 expressing neurons would modulateitch related behaviors. To selectively target Tacr1-­expressing neurons in the RVM, we employed achemogenetic approach. AAV-­DIO-­hM3dq-­mCherry was injected into the RVM of Tacr1 cre mice,

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Figure 2. In Tacr1 cre +/-­mice receiving AAV-­DIO-­hM3Dq-­mCherry injections in the RVM, mCherry strongly colocalized with Tacr1 expression. (A–C): Images of RVM cells expressing mCherry (A), anti-­Tacr1 antibody (B), and double-­labeled cells (C). (D): 84% of mCherry expressing neurons co-­express Tacr1. N=7. Scale bar (25 µm
The online version of this article includes the following source data for figure 2:
Source data 1. Data for co-­expression of DREADDs to Tac1r.

resulting in selective expression of hM3Dq in Tacr1-­expressing neurons (84% of RVM mCherry labeledneurons also expressed Tacr1 Figure 2A–D).

Activation of RVM Tacr1 expressing neurons inhibits acute itch
To test the functional role of the RVM Tacr1-­expressing population of neurons in itch modulation,
we used DREADDs (hM3dq) to activate these neurons during pruritogen-­evoked scratching behaviors (Figure 3A). Following viral injection, we observed clear expression of mCherry in the RVM(Figure 3B). Activation of DREADD-­expressing RVM neurons in Tacr1 cre mice using clozapine significantly attenuated pruritogen-­evoked scratching behavior. After clozapine administration (0.01 mg/
kg, ip; Gomez et al., 2017), there was a significant reduction in scratch bouts elicited by intradermal
histamine as compared to systemic administration of saline vehicle (Figure 3C). There was a significantly stronger inhibition of histamine-­evoked scratching behavior in male compared to female mice
(Figure 3—figure supplement 3e). Similarly, there was a significant reduction in chloroquine-­evoked
scratching (Figure 3D). In contrast, clozapine administration had no significant effect on the withdrawal threshold to mechanical von Frey stimuli (Figure 3E) or withdrawal latency to thermal stimulation (Figure 3F). Administration of clozapine in control vector (Tacr1-­mCherry) mice had no significant
effect on scratching behavior elicited by intradermal injection of histamine (Figure 3C) or chloroquine(Figure 3D). Likewise, clozapine administration had no significant effect on mechanical (Figure 3E)or thermal hindpaw withdrawals in vector controls (Figure 3F). Finally, neither a low (0.01 mg/kg)
nor a higher dose (0.1 mg/kg) of clozapine had any significant effect on histamine- or chloroquine-­
evoked scratching behavior or on mechanically- or thermally-­evoked paw withdrawals in wildtype
mice (Figure 3—figure supplement 2).
Our results were independently confirmed using a separate line of Tacr1-­CreER mice (Huang
et al., 2016), which received intra-­RVM microinjection of the excitatory DREADD AAV2-­DIO-­hM3dq-­
mCherry. Chemogenetic activation using CNO significantly reduced chloroquine evoked scratch bouts(Figure 3—figure supplement 1A) and spontaneous scratching behavior (Figure 3—figure supplement 1B), and also significantly reduced the von Frey mechanical withdrawal threshold (Figure 3—
figure supplement 1C) with no effect on thermal withdrawal latency (Figure 3—figure supplement
1D). CNO had no effect in the control vector mice. Independent experiments in two lines of Tacr1
cre mice support the conclusion that activation of RVM Tacr1 expressing neurons inhibits pruritogenevoked scratching behavior.

RVM Tacr1 expressing neurons inhibit chronic itch
Since activation of RVM Tacr1-­
expressing neurons inhibited acute pruritogen-­evoked scratching
behavior, we next wanted to determine whether chronic itch related behaviors would be affected.
We used the imiquimod model of psoriasisiform dermatitis in Tacr1 cre mice receiving intra-­RVM

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Figure 3. Chemogenetic activation of RVM Tacr1 expressing neurons inhibits itch related behavior. (A) AAV5-­DIO-­hM3Dq-­mCherry or AAV5-­DIO-­
mCherry was injected into the RVM of Tacr1 cre mice. (B) Expression of hM3Dq-­mCherry was limited to the RVM. Gt: gigantocellularis; RMg: raphemagnus; RPa: raphe pallidus. Scale bar (1 mm). (C) Administration of clozapine caused a significant reduction in histamine-­evoked scratching in hM3Dq
expressing mice (7 males, 7 females, M = (59.32; 14.75), t=3.715, DF = 13, 95% CI [18.65, 70.49], p=0.0026), but not in control vector mice (six males,
three females, M = (67.28; 60.61), t=0.6235, DF = 8, 95% CI [–24.11, 37.44], p=0.6235). (D) Administration of Clozapine caused a significant reduction inchloroquine-­evoked scratching in hM3Dq mice (seven males, eight females, M = (163.2; 54.33), t=4.72, DF = 14, 95% CI [59.40, 158.3], p=0.0003) but notin control vector mice (six males, four females, M = (97.90; 112.9), t=0.4129, DF = 9, 95% CI [–23.45, 36.78], p=0.4129). (E) Clozapine administration did
not significantly change mechanical withdrawal thresholds in hM3Dq (seven males, eight females, M = (5.549; 5.610), t=0.1309, DF = 14, 95% CI [–1.060,
0.9378], p=0.8977) or control vector mice (six males, four females, M = (4.742; 4.958), t=0.6342, DF = 9, 95% CI [–0.9867, 0.5546], p=0.5418). (F) Clozapine
administration did not significantly change thermal withdrawal thresholds in hM3Dq (seven males, eight females, M = (4.735; 5.587), t=1.262, DF =14, 95% CI [–2.300, 0.5961], p=0.2276) or control vector mice (six males, four females, M = (3.718; 3.737), t=0.0597, DF = 9, 95% CI [–0.7260, 0.6886],
p=0.9537). (C–F) *p<0.05, **p<0.01, ***p<0.001, Students paired t-­test.
The online version of this article includes the following source data and figure supplement(s) for figure 3:
Source data 1. Behavioral data of RVM Tac1r DREADDs activation with clozapine.
Figure supplement 1. Chemogenetic activation of RVM Tacr1 expressing neurons inhibits itch-­related behavior.
Figure supplement 1—source data 1. Behavioral data of RVM Tac1r DREADDs activation with CNO.
Figure supplement 2. Clozapine administration does not affect acute itch or pain behavior.
Figure supplement 2—source data 1. Data for Clozapine administration effects on behavior.
Figure supplement 3. Sex differences.
Figure supplement 3—source data 1. Data for behavioral sex differences.

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injection of hM3Dq or control vector. Mice were treated daily with topical application of 5% imiquimod
cream for 5 days. This resulted in a significant increase in spontaneous scratching on treatment day
3 (Figure 4A, dark blue) and significantly increased alloknesis scores on treatment days 1, 3, and 5(Figure 4D, dark blue). Application of vehicle (Vanicream) had no effect on spontaneous scratchingor alloknesis (Follansbee et al., 2019). Following administration of clozapine on day 5, there was asignificant reduction in the number of spontaneous scratch bouts (Figure 4A, light blue). Figure 4B
shows that scratch bouts in individual animals were significantly reduced after the administrationof clozapine. Clozapine administered on days 3 and 5 also significantly reduced alloknesis scores(Figure 4D, light blue). Figure 5E shows significant reductions in alloknesis scores of individual animals
after as compared to before clozapine. Mice that received intra-­RVM microinjection of control vector
also showed significant increases in spontaneous scratching (Figure 4A, dark green) and alloknesisscores (Figure 4D, dark green) following imiquimod treatment. Administration of clozapine did not
significantly affect spontaneous scratching (Figure 5C) or alloknesis scores (Figure 5F) in these mice.
These results show that activation of RVM Tacr1-­expressing neurons reduces itch related behaviors ina model of chronic itch in addition to acute pruritogen-­evoked scratch bouts.

RVM ON cells express Tacr1
Previous reports had shown, in rats, that RVM ON cells were facilitated by local microinjection of SP(Budai et al., 2007; Zhang and Hammond, 2009) and ablation of RVM neurons which express Tacr1,
was antihyperalgesic (Khasabov et al., 2017a), consistent with a pronociceptive role for RVM ON
cells.
We used Tacr1 cre mice which were injected with an AAV encoding channelrhodopsin (AAV5:DIO-­
ChR2-­eYFP). Four weeks later, the mice were anesthetized with sodium pentobarbital for single-­unit
recording with a microelectrode whose tip extended a few hundred micrometers beyond the tip ofan optic fiber it was affixed to (Figure 5A). Injected mice exhibited robust expression of eYFP in the
RVM (Figure 5B). ON and OFF cells were identified based on their response to a pinch stimulus. Once
identified, blue light (473 nm, 0.25–5 mW) was applied and the cell was tested for entrainment to
the light stimulus. Figure 5C shows an example of an ON cell in RVM that responded to pinch prior
to the onset of EMG activity and was faithfully entrained to 5 hz light stimulation (Figure 5D). This
neuron received a total of 37 light pulses at 2 hz and fired 32 action potentials within 20ms of thelight onset (Figure 5E) with a calculated efficiency index of 0.86 (see below), and an average latencyof 8.14ms. The latencies measured presently compare favorably with those reported previously for
hippocampal neurons (Zhang et al., 2013). The latency, or on rate, of neuronal activation differs bycell type (Herman et al., 2014) and increases with diminishing light density (Lin et al., 2009). Out of
22 identified ON cells, 17 were entrained to the light stimulus, 1 was inhibited and 4 were not affected(Figure 5F). It is possible that our estimate is an under representation since viral transduction was notcompletely efficacious. At the end of the recording a lesion was produced which we histologically verified (Figure 5J). Since the majority of RVM ON cells were entrained to light stimulation, we conclude
that the majority of ON cells likewise express Tacr1.
While our primary interest was RVM ON cells, we wanted to determine whether Tacr1 is also
expressed in other cell types, such as RVM OFF cells. During our recordings we often found RVM OFF
cells. Out of 14 identified OFF cells, none were directly activated by the light stimulus, with 7 showing
a clear inhibitory response to the light stimulation while the other 7 were unaffected (Figure 5F).
Often the degree of inhibition of the OFF cell increased proportionately to the stimulation frequency(Figure 5G–I). These results support the hypothesis that RVM ON cells provide an inhibitory input
onto RVM OFF cells (Fields and Heinricher, 1989) and may underlie the RVM OFF cell mediatedpause during noxious stimulation.
Occasionally, Neutral cells were identified by their response to optic stimulation (n=3), and each
was strongly entrained (Figure 5F). Since Neutral cells are numerous, not affected by noxious stimulation and their contribution, if any, to descending modulation is unknown, they were not investigatedfurther. We thus conclude that while most presently-­recorded light-­sensitive cells were ON cells, othercell types including Neutral cells also expresses Tacr1.
To validate our results from optotagging, we used several metrics to determine whether a light-­
sensitive neuron was truly entrained to the optic stimulus. We assumed that each optic stimulus was
likely to directly excite a neuron expressing ChR2 and tested this in different ways. An efficiency index

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Figure 4. Chemogenetic activation of RVM Tacr1 neurons reduces spontaneous scratching and alloknesis in the imiquimod model of chronic
psoriasisiform itch. (A) Application of imiquimod (1%, 0.05 g, Taro) once per day produced a significant increase in spontaneous scratching at day 3
for both DREADDs (dark blue, RM ANOVA, Dunnett’s multiple comparisons Day0 vs Day 3, q=0.18511, p=0.0234) and control vector (dark green, RM
ANOVA, Dunnett’s multiple comparisons Day0 vs Day 3, q=2.549, p=0.0382) mice. Following clozapine, there was a significant reduction in spontaneous
scratching on day 5 for the DREADDs mice (dashed light blue, Paired t test n=8, M = (17.25; 5.250), t=2.38, DF = 7, 95% CI [-23.92,–0.07597], p=0.0489)
but no significant change in control vector mice (dashed light green, Paired t test n=10, M = (36.75; 49.56), t=1.343, DF = 9, 95% CI [–35.38, 9.752],
p=0.2213). (B) Graph shows individual DREADDs animals’ spontaneous scratch bouts pre- and post-­clozapine. Following clozapine there was asignificant reduction in scratching. Blue: individual counts; black: mean +/-SEM. Paired t test n=8, M = (17.25; 5.250), t=2.38, DF = 7, 95% CI [-23.92,–
0.07597], p=0.0489 (C) Graph as in B for mice in vector control group, in which clozapine had no significant effect. Green: individual counts; black: mean+/-SEM. Paired t test n=10, M = (36.75; 49.56), t=1.343, DF = 9, 95% CI [–35.38, 9.752], p=0.2213 (D) Imiquimod induced significant increases in alloknesisscores on days 3 and 5 of treatment in DREADDs (dark blue, RM ANOVA, Dunnett’s multiple comparisons, Day0 vs Day 3, q=9.379, p=0.<0.0001, Day0
vs Day5, q=4.00, p=0.0129) and vector control (dark green, RM ANOVA, Dunnett’s multiple comparisons, Day0 vs Day 3, q=4.811, p=0.0025, Day0
vs Day5, q=5.014, p=0.0019) mice. Following clozapine administration on days 3 and 5 there were significant reductions in alloknesis scores for the
DREADDs mice (dashed light blue, Paired t test n=8, M = (2.250; 0.6250) t=6.177, DF = 7, 95% CI [1.003, 2.247], p=0.0005) but not control vector groups(dashed light green, Paired t test n=10, M = (1.300; 1.400) t=0.3612, DF = 9, 95% CI [–0.7264, 0.5264], p=0.7263). (E) Clozapine resulted in a significant
reduction in alloknesis scores (format as in B), Paired t test n=8, M = (2.250; 0.6250) t=6.177, DF = 7, 95% CI [1.003, 2.247], p=0.0005 (F) Clozapine had no
Figure 4 continued on next page

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Figure 4 continued

effect on alloknesis scores in vector controls (format as in C). Paired t test n=10, M = (1.300; 1.400) t=0.3612, DF = 9, 95% CI [–0.7264, 0.5264], p=0.7263.
Paired students t-­test *p<0.05, **p<0.01, ***p<0.001. (A–F) n=4–5 males, 4–5 females/group.
The online version of this article includes the following source data for figure 4:
Source data 1. Data for RVM Tac1r activation in psoriasis model of chronic itch.

was calculated to determine whether a neuron was entrained to the optic stimulus, by counting thenumber of evoked action potentials divided by the number of optic stimuli (Figure 5—figure supplement 1). The 3 Neutral cells had the highest efficiency index (>1), which was due to occasional firingof ‘doublet’ action potentials in response to the optic stimulus (Figure 5—figure supplement 1C, D).
ON cells classified as light sensitive also had a high efficiency index, which decreased with increasing
optic stimulation frequency most likely due to desensitization of ChR2. We additionally assumed thatthe neuronal firing rate would reflect the number of optic stimuli. Indeed, the neuronal firing rate inlight sensitive neurons increased with the stimulation frequency, while non-­light sensitive neurons
showed no change (Figure 5—figure supplement 2). In neurons which were inhibited by optic stimulation, there was a decrease in firing rate following light stimulation (Figure 5—figure supplement
2, OFF cells). Finally, we assumed that the light-­evoked latency of neuronal action potentials would
be consistent. For the population of light-­sensitive ON cells, the average latency of responses to theoptic stimuli delivered at 2 hz was 7.6 (+/-1.12) ms (Figure 5—figure supplement 3). Thus, theseexperiments strongly support the hypothesis that the majority of RVM ON cells express Tacr1.

Discussion
Previous studies indicate a pronociceptive role for SP acting at Tacr1 expressing neurons in RVM(Budai et al., 2007), many of which have descending spinal projections (Pinto et al., 2008). A novel
finding of the present study is that pharmacological and chemogenetic activation of Tacr1-­expressing
RVM neurons inhibited itch-­related scratching behavior, with mild mechanical sensitization and noeffect on thermal nociception. Our optotagging experiments provide evidence that the majority of
ON cells express Tacr1. These results argue for a causal role of a subpopulation of Tacr1-­expressing
ON cells in the inhibition of itch-­related scratching behavior, in contrast to their pronociceptive role.

Role of SP in RVM in descending modulation of pain and itch
SP (Ljungdahl et al., 1978) and Tacr1 (Saffroy et al., 2003) are present within the RVM. SP acting in
the RVM potentiated ON cells, induced pronociceptive behavioral effects and sensitized spinal wide
dynamic range dorsal horn neurons (Budai et al., 2007; Khasabov et al., 2017b). SP appears not tobe released in the RVM in the absence of injury, but is released under inflammatory conditions elicited by CFA or capsaicin to induce Tacr1-­dependent hyperalgesia (Brink et al., 2012; Hamity et al.,2010; Khasabov et al., 2017a). The present study expands on this work in important ways. Firstly,
we confirm that SP potentiates noxious pinch-­evoked responses of mouse RVM ON cells. Secondly,
our optotagging data provide evidence that the Tacr1-­expressing neurons are ON cells. Importantly,
our results support a role for these neurons in the inhibition of acute and chronic itch. Intra-­RVM
microinjection of SP or chemogenetic activation of Tacr1-­expressing neurons in two different Tacr1
cre lines significantly decreased pruritogen-­evoked as well as chronic itch related scratching behavior,
while facilitating mechanical nociception in most experiments with no effect on thermal nociception.
This latter observation contrasts with a previous report that intra-­RVM microinjection of an SP agonist
enhanced thermal nociceptive behavior in rats (Khasabov et al., 2017b) a discrepancy that might be
attributed to a species difference.

Descending modulation of itch
Optogenetic activation of GABAergic neurons in RVM facilitated mechanical, but not thermal nociception in mice (François et al., 2017), consistent with our data. The latter authors suggested that the
GABAergic RVM neurons might represent ON cells, which descend to presynaptically inhibit mechanonociceptor input onto spinal inhibitory enkephalinergic interneurons that in turn contact the spinal

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Figure 5. Optotagging of RVM Tacr1 neurons. (A) RVM cells were recorded with a microelectrode coupled to an optic fiber. (B) Injection of AAV-­ChR2-­
eYFP in the RVM of Tacr1 cre+/- mice caused strong expression of eYFP. Scale bar (100 µm). (C) ON cells were identified based on their pinch-­evoked
response that preceded the hindlimb withdrawal as monitored by EMG in biceps femoris. (D) Cells identified as RVM ON cells were optically stimulated(5 mW, 472 nm). This neuron faithfully responded to each pulse in a 5 hz train. (E) Light entrainment was analyzed by creating peristimulus-­time
Figure 5 continued on next page

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Figure 5 continued

histograms (PSTH) of action potentials that occurred within a 20 ms window following the onset of each light pulse. This neuron responded consistently
at a latency of approximately 8.14 ms with a calculated efficiency index of 0.86. (F) Distribution of RVM ON, OFF, and Neutral cells which were excited(red), inhibited (blue), or not affected (yellow) by optic stimulation. (G) Application of a pinch stimulus elicited a hindlimb withdrawal (dotted line) and apause in firing that is typical of OFF cells. (H) There was an intermittent decrease in OFF cell firing during 5 hz optic stimulation and a (I) total cessationof firing during 20 hz optic stimulation. (J) Lesion sites from the optotagging recordings of RVM ON (red circles), OFF (blue triangles), and NEUTRAL
cells (green squares). Numbers to right indicate Bregma coordinates.
The online version of this article includes the following source data and figure supplement(s) for figure 5:
Figure supplement 1. Efficiency index of classified RVM neurons.
Figure supplement 1—source data 1. Data for efficiency index analsyis of optotagging experiments.
Figure supplement 2. Firing rates of classified RVM neurons in response to optic stimulation.
Figure supplement 2—source data 1. Data for firing rate analsyis of optotagging experiments.
Figure supplement 3. Response latency of ON cells to optic stimulation.
Figure supplement 3—source data 1. Data for response latency of optotagging experiments.

mechanonociceptive pathway. Thus, activation of the descending GABAergic neurons would facilitate
mechanical nociception via disinhibition.
Our study shows that RVM Tacr1 neurons represent a population of RVM ON cells, which when
activated, reduce pruritogen-­evoked scratching. A limitation of this study is that we were not able
to directly demonstrate that inhibition of spinal pruriceptive transmission caused the reduction in
scratching behavior. Previous studies have shown that 31% of functionally identified RVM ON cells(Vanegas et al., 1984), and 42.5% of Tacr1-­expressing RVM neurons (Pinto et al., 2008), project tothe spinal cord, supporting the descending modulation of spinal itch processing. Moreover, recent
studies report that activation of spinal projection neurons originating in the locus coeruleus (Kogaet al., 2020) or somatosensory cortex (Wu et al., 2021) suppress itch-­related scratching behavior.
Given this and the historical evidence of the spinal action of RVM ON cells, the most parsimonious
explanation is that RVM Tacr1-­expressing neurons with descending axons exert an inhibitory effecton spinal pruriceptive transmission to reduce scratching behavior. However, we cannot exclude thepossibility that Tacr1-­expressing RVM neurons lacking spinal projections exert an antipruritic effect via
an unknown supraspinal action.
It was recently reported that activation of neurons in RVM that express the G-­protein-­coupled
estrogen receptor (GPER) suppresses signs of acute and chronic itch (Gao et al., 2021). It is currently
not known if GPER-­expressing neurons co-­express Tacr1.
Electrical stimulation of the PAG inhibited nocifensive behavior in rats (Mayer et al., 1971; Reynolds, 1969), and the PAG projects directly to the RVM (Behbehani and Fields, 1979). Activation of
PAG GABAergic neurons, and inhibition of glutamatergic neurons, reduced scratching behavior underboth acute and chronic itch conditions (Samineni et al., 2019) but facilitated nocifensive behaviors(Samineni et al., 2017). Activation of Tac1 (SP-­expressing) neurons in the PAG was shown to facilitate
pruritogen-­evoked scratching via release of glutamate onto neurons in the RVM (Gao et al., 2019).
In contrast, our results indicate that activation of RVM Tacr1 neurons inhibited pruritogen-­evoked
scratching behavior. This potentially represents a difference in the synaptic connections of PAG Tac1-­
expressing neurons with glutamate- and/or Tacr1-­expressing RVM neurons. Previous studies havereported projections to RVM from SP-­expressing neurons located in dorsolateral PAG, dorsal raphe
nucleus, cuneiform nucleus, and lateral hypothalamus (Chen et al., 2013; Holden and Pizzi, 2008;
Yin et al., 2014). It is thus possible that Tacr1-­expressing RVM neurons that inhibit scratching are
activated by different SP inputs than the RVM neurons activated by PAG TAc1-­expressing neurons
that facilitate scratching.

Modality specific role of RVM ON cells
For decades, the role of RVM ON cells has been considered faciliatory for spinal nociceptive transmission. Our results suggest that the role of RVM ON cells is modality specific, with RVM ON cells facilitating spinal nociceptive while inhibiting pruriceptive transmission. Pruritogens and algogens similarly
excited RVM ON and inhibited OFF cells, implying that the opposing modulatory effects of ON cells
on spinal nociceptive vs. pruriceptive transmission likely occurs via separate spinal circuits. RVM ON

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cells have a GABA-­mediated inhibitory connection to spinal enkephalinergic neurons (François et al.,2017), raising the possibility that spinal enkephalinergic tone accounts for the opposing effects on
spinal nociceptive and pruriceptive transmission. Since pain suppresses itch, we cannot exclude thepossibility that activation of spinal pronociceptive circuitry inhibits itch transmission.

Treatment of chronic itch with Tacr1 antagonists
The Tacr1 antagonists aprepitant and serlopitant were recently shown to be partially effective in
reducing chronic itch of various etiologies. Tacr1 antagonists might act at Tacr1 that is expressed byspinothalamic and spinoparabrachial neurons (Yang et al., 2021 & Todd, 2010) to reduce pruriceptive
transmission. Indeed, ablation of Tacr1-­expressing spinal and medullary neurons reduces pruritogen-­evoked scratching behavior (Carstens et al., 2010). The present results suggest that Tacr1 antagonists also have an opposite effect to block Tacr1-­mediated descending inhibition of itch. Systemic
administration of Tacr1 antagonists thus appear to have offsetting supraspinal and spinal effects,
potentially explaining the limited efficacy of Tacr1 antagonists for chronic itch.

Key resources table

Materials and methods
Additional
information

Reagent type (species) or resource Designation

Source or reference

Identifiers

Strain, strain background (M.
musculus)

Tacr1 creDong Lab

Strain, strain background (M.
musculus)

Tacr1 creER

Dong Lab (Huang et al., 2016)

Strain, strain background (AAV)

aav5-­hSyn-­DIO-­hM3Dq-­mCherry

Addgene44361-­AAV5

Strain, strain background (AAV)

aav5-­hSyn-­DIO-­mCherry

Addgene50459-­AAV5

Strain, strain background (AAV)

aav5-­Ef1a-­hChR2-­mCherry

Addgene20297-­AAV5

Antibody

Mouse monoclonal: anti-­Tacr1
conjugated 488

SCBT

sc-­365091 AF488

Chemical compound, drug

Substance P (SP)Tocris1156/5Chemical compound, drug

Histamine Dihydrochloride (HA)

Sigma-­Aldrich

H7250

Chemical compound, drug

Chloroquine Diphosphate (CLQ)

Sigma-­Aldrich

C6628

Chemical compound, drug

Pentobarbital Sodium

Sigma-­Aldrich

P3761

Chemical compound, drug

Buprenorphine Hydrochloride

Amerisoucebergen

NDC42023-­179-­05

Software, algorithm

5 Prism

Graphpad(1:50)

Animals
Experiments were performed using wild type mice (c57BL/6 j, Jackson Labs, Bar Harbor ME), Tacr1
cre+/- (courtesy of Dr. X. Dong, Johns Hopkins University), and Tacr1 creER +/-­mice (Huang et al., 2016)of both sexes, 8–10 weeks of age, all on a C57Bl/6 background. Mice were given free access to foodand water and housed under standard laboratory conditions. Mice were housed in-­lab and at the
animal housing facility, with a natural light cycle and 12 hr light dark cycle, respectively. Mice wereallowed to habituate for at least 3 days following transfer from the animal facility to lab housing before
use in behavioral experiments. Mice were cohoused with between 1 and 4 mice/cage. All procedures
were approved by the University of California, Davis and University of Pittsburgh Animal Care and Use
Committees and followed the ARRIVE guidelines to the extent possible.

Pharmacologic agents
Clozapine was dissolved in saline and administered intraperitoneally (ip) at concentrations of 0.01 and
0.1 mg/kg. Clozapine-­N-­oxide (CNO;Tocris, Bristol UK) was dissolved in phosphate-­buffered saline andadministered ip (5 mg/kg). In clozapine and CNO treated mice, experiments were conducted 30 min
following their administration. Histamine HCl (Sigma, St. Louis MO; 0.5% in 10 µl) or chloroquine

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diphosphate salt (Sigma; 1% in 10 µl) were dissolved in physiological saline and administered intradermally via a 30 g needle in the nape of the neck. Imiquimod cream (5%; Aldara, 50 mg; 3 M Health Care
Limited, UK) was administered topically once per day to shaved skin on the rostral back for 5 days.

Stereotaxic injections and cannula implantation
Animals were anesthetized with 2% isoflurane and placed in a stereotaxic head frame. A burr hole
was made in the calvarium and a Hamilton microsyringe loaded with virus was stereotaxically placedsuch that the tip was at the injection site in RVM (RC: –5.5 to –5.8 mm, ML: 0.0, DV: –4.2 to –6 mm).
Virus (0.25–1 µl) containing either AAV5: hSyn-­DIO-­hM3Dq-­mCherry (excitatory DREADD; Addgene,
Watertown MA), AAV5: hSyn-­DIO-­mCherry (control vector, Addgene), or AAV5: hSyn-­DIO-­ChR2-­eYFP(Addgene) was injected into the RVM. Virus was infused at an approximate rate of 100 nL/min. The
injection needle was left in place for an additional 15 min post-­injection and then slowly withdrawn.
The incision was closed using Vetbond and animals were given ketofen (10 mg/kg ip) or buprenorphine (0.05 mg/kg ip) and allowed to recover on a heating pad. A 4-­week recovery period ensued
prior to experimentation.
For intracranial drug injections, an injection cannula (Plastics One, Roanoke VA) was implanted
stereotaxically with the tip targeted 1–2 mm above the RVM. For optogenetic stimulation, following
microinjection of AAV5: hSyn-­DIO-­ChR2-­eYFP into RVM, an optic fiber (Doric Lenses, Quebec Canada)
was stereotaxically implanted at the target site. Dental cement was used to affix the injection cannula
or optic fiber to the skull. Post-­implantation mice received buprenorphine (0.05 mg/kg ip). Mice recovered for at least 4 weeks prior to experimentation.

Intracranial drug microinjection
Mice were habituated for 15 min prior to intracranial drug injection in clear cylindrical glass enclosuresand videotaped from above. Saline (0.5 µl) or Substance P (SP, 0.5 µl, 10 nmol, Tocris, Minneapolis
MN) was microinjected. Fifteen minutes post-­injection mice were tested for itch or pain behaviors as
described below. Both male and female mice were used.

Chemogenetics
Thirty minutes after ip administration of saline, clozapine or CNO, behavioral testing commenced. We
verified the efficacy of clozapine by observing an increase in cfos staining in the RVM of DREADDs
versus control vector mice as well as saline versus clozapine (data not shown).

Behavior
Scratching behavior: Scratch bouts were defined as back-­and-­forth hindpaw movements directed to
the rostral back, followed by biting the toes and/or placement of the hindpaw on the floor. Scratchbouts elicited by histamine, chloroquine or saline vehicle were videotaped and counts made by atleast two blinded observers. Nociceptive behavioral assays (von Frey; Hargreaves) were conducted byinvestigators blinded as to treatment. Successive behavioral tests were conducted in pseudorandom
order and spaced 1 week apart.
Von Frey: Mechanical sensitivity was measured in two ways. Mice stood on a mesh floor allowing
access to the plantar surface from below. Using the Chaplan up-­down method (Chaplan et al., 1994),
calibrated von Frey filaments (North Coast Medical Inc) were applied to the plantar surface. Paw
lifting, shaking, and licking were scored as positive responses. Averaged responses were obtained
from each hindpaw, with 3 min between trials on opposite paws, and 5 min between trials on the same
paw. Alternatively, the force at the moment of hindpaw withdrawal was measured using an electronic
von Frey device (2390; IITC, Woodland Hills CA). Measurements were again taken from each hindpaw
with 3 and 5 min between trials on the same or opposite paw.
Hargreaves: Animals were acclimated on a glass plate held at 30 °C (Model 390 Series 8, IITC
Life Science Inc). A radiant heat source was applied to the hindpaw and latency to paw withdrawal
was recorded (Hargreaves et al., 1988). Two trials were conducted on each paw, with at least 5 min
between tests of opposite paws and at least 10 min between tests of the same paw. To avoid tissue
damage, a cut off latency of 20 s was set. Values from both paws were averaged.

In vivo single-unit recording
Adult mice (6 weeks) mice were anesthetized with pentobarbital sodium (60 mg/kg, ip). The head was
secured in a stereotaxic frame and an opening was made in the occipital bone. The animal’s body

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temperature was maintained with a heating pad and external heating source. Teflon coated silver
wires were inserted into the biceps femoris to record electromyographic (EMG) activity. A single-­unit
recording microelectrode (10 MOhm, Frederick Haer Inc, Bowdoin ME) was coupled to a 33-­gauge
injection cannula such that the tip of the recording microelectrode extended several hundred μm
beyond the tip of the injection cannula and was inserted into the RVM. ON cells were identified bya hindpaw pinch-­evoked increase in firing that preceded the hindlimb withdraw reflex measured as
EMG activity from fine wires inserted in the biceps femoris. Once an ON cell was identified, either
saline or SP (0.5 μl, 10 nmol) was microinjected and changes in firing rate to repeated consistent pinch
stimuli were recorded. Responses to pinch stimuli were normalized to the initial response for each
unit. Electrode voltages were amplified and digitized (CED 1401, CED, Cambridge UK) and analyzed
with Spike2 (CED). At the end of the recording, a lesion was produced at the last recording site bypassing direct current through the microelectrode, the brain was harvested postmortem and postfixedin 10% formalin.

Optotagging
At least 4 weeks following injection of AAV5: hSyn-­DIO-­ChR2-­eYFP into RVM, single-­unit recordings
were made from RVM as described above using a Tungsten microelectrode attached to an optic
fiber such that the microelectrode tip extended a few hundred microns beyond the optic fiber. In
most experiments ON cells were functionally characterized as described above, and OFF cells werecharacterized by a pinch-­evoked pause in ongoing activity that preceded the withdrawal reflex. ON
and OFF cells were then tested for entrainment to optic stimulation at 473 nm wavelength and 0.25
mW - 5 mW light output (5–20 Hz, 10 ms pulse duration) from a laser (Laserglow R471003GX). In someexperiments, optic stimulation was used as to isolate light-­sensitive neurons, which were then identified as ON, OFF or Neutral based on their response to pinch. Efficiency indices were calculated as
the number of action potentials firing within a 20 ms window following the onset of the optic stimulus
divided by the total number of optic stimulus pulses, and were used to determine whether a neuron
was entrained to the optic stimulus. Both male and female mice were used for all electrophysiologicalexperiments.

Imiquimod treatment
Imiquimod cream was applied topically to the shaved area on the rostral back once per day for 5
consecutive days. Treatment groups consisted of age-­matched male and female NK-­1-­
cre micethat had received intra-­RVM injection of AAV5: DIO-­hM3Dq-­mCherry or the control vector AAV5:
hSyn-­DIO-­mCherry. Imiquimod treatment induced signs of skin pathology including skin scaling anderythema. As measures of chronic itch, we assessed spontaneous scratching, and alloknesis, 23 hrfollowing imiquimod treatment. Mice were videotaped and tested between 10 AM and 5 PM, witheach individual mouse tested at the same time each day. The mice were habituated to glass cylinders
for 3 successive days prior to recording. Animals were videotaped from above for 30 min. Behavioral videos were analyzed by two blinded observers. Only discrete bouts of spontaneous hindlimb
scratches directed towards the application site were counted, as described previously (Akiyama
et al., 2016) and summed over the 30 min period. Alloknesis was assessed as previously described(Akiyama et al., 2012). The mouse was placed in an enclosed area and a 0.07 g von Frey monofilament was applied to the perimeter of the imiquimod application area five consecutive times. The
alloknesis score consisted of the number of immediately occurring hindlimb scratch bouts directed tothe stimulus site.

Immunofluorescence
Four weeks after intra-­
RVM injection of AAV5:hSyn-­
DIO-­
hM3Dq-­
mCherry or the control vector(AAV5:hSyn-­DIO-­mCherry) in Tacr1 cre mice, clozapine (0.01 mg/kg, i.p.) was injected, followed 1 hrlater by perfusion (4% paraformaldehyde), harvesting of brains and post-­fixation overnight in 4% paraformaldehyde. Brains were sectioned (20 μm) on a freezing microtome and stained as free floating
sections. DREADDs-­expressing neurons were counterstained for Tacr1 expression with a conjugatedTacr1 antibody (D-­11, sc-­365091, Santa Cruz Biotechnology, Dallas TX) at 1:50 overnight at 4 °C.
mCherry expressing neurons were identified in the RVM and then observed for Tacr1 expression. All
slides were mounted with vectashield and imaged with confocal microscopy. Staining intensity was

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measured relative to the red fluorescence (from DREADDs) and was quantified using FIJI (Schindelin
et al., 2012).

Statistical analysis
All statistical analyses were performed using GraphPad Prism. Values are presented as mean +/-SEM.
Statistical significance was assessed using students t-­test or a two-­way, repeated measures ANOVA
with Bonferroni’s correction, unless otherwise specified. Significance was indicated by p<0.05. Sample
sizes were based on pilot data and are similar to those typically used in the field.
For analysis of effects of intracranial microinjections, a paired students t-­test was used to compare
the effects of intracranial injection of saline versus SP on behavioral measures. Two-­way ANOVA determined a lack of significant sex x SP microinjection interaction, so the data from both sexes werepooled. For chemogenetic experiments, a paired t-­test was used to compare scratch counts and nociceptive measures following vehicle vs. clozapine or CNO injection, or between vector controls and
DREADDs mice following clozapine or CNO. For optogenetic experiments a paired t-­test was similarly
used to compare behavioral measures during and in the absence of optic stimulation.
For experiments with imiquimod, paired students t-­tests compared the effects of ip injectionof saline versus clozapine on spontaneous scratch bouts and alloknesis scores. A two-­way ANOVA
revealed no interaction for sex x clozapine administration, so the data were pooled for further analysis.
For single-­unit recordings, a two-­way repeated measures ANOVA with bonferroni post-­hoc test
was used to compare the neuronal responses to pinch following intra-­RVM injection of saline or SP for
60 min post-­injection.
For immunohistochemical staining for c-­fos, students t-­test compared the staining intensity of c-­fos
and the proportion of c-­fos-­positive neurons in Tacr1-­expressing neurons following administration of
clozapine, in mice receiving DREADDs or control vector injections.

Acknowledgements
We thank Dr. Dong (Johns Hopkins University) for the generous donation of Tacr1-­cre mice.

Additional information
Funding
FunderGrant reference number

AuthorNational Institutes of
HealthAR076434

Earl Carstens

National Institutes of
HealthAR057194

Earl Carstens

National Institutes of
Health

AR063772

Sarah E Ross

National Institutes of
HealthNS096705

Sarah E Ross

National Institutes of
HealthF31NS113371

Eileen Nguyen

National Institutes of
HealthT32GM008208

Eileen Nguyen

The funders had no role in study design, data collection and interpretation, or thedecision to submit the work for publication.

Author contributions
Taylor Follansbee, Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing; Dan Domocos, Formal analysis, Funding acquisition, Investigation;
Eileen Nguyen, Amanda Nguyen, Lauren Velasquez, Data curation, Investigation; Aristea Bountouvas,

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Investigation; Mirela Iodi Carstens, Resources, Supervision, Investigation, Methodology, Project
administration; Keiko Takanami, Data curation, Formal analysis, Investigation; Sarah E Ross, Supervision, Writing – review and editing; Earl Carstens, Conceptualization, Data curation, Formal analysis,
Supervision, Funding acquisition, Methodology, Writing – original draft, Writing – review and editing
Author ORCIDs
Taylor Follansbee ‍ ‍http://orcid.org/0000-0002-4470-9324
Sarah E Ross ‍ ‍http://orcid.org/0000-0003-2593-3133
Earl Carstens ‍ ‍http://orcid.org/0000-0003-4696-8116
EthicsThis study was performed in accordance with our approved IACUC protocol (21316) at the Universityof California, Davis.
Decision letter and Author response
Decision letter https://doi.org/10.7554/eLife.69626.sa1
Author response https://doi.org/10.7554/eLife.69626.sa2

Additional files
Supplementary files•  Transparent reporting form
Data availability
All data generated or analyzed during this study are included in the manuscript and supporting files.

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